Analytical process

ABSTRACT

KNOWN ROTATING SPECTROPHOTOMETERS HAVE A SERIES OF CUVETS ARRANGED CONCENTRICALLY AROUND A HORIZONTALLY ROTATABLE DISC SO THAT THE DISC IS ROTATED, CENTRIFUGAL FORCE MIXES AND TRANSFERS REAGENTS AND SAMPLES TO THE CUVETS. AS EACH CUVET PASSES A LIGHT SOURCE, THE ABSORBANCE OF EACH INDIVIDUAL SAMPLE IS DETECTED AND MEASURED PHOTOMETRICALLY AND AN ELECTRICAL SIGNAL PROVIDED WHICH IS INDICATIVE OF THE EXTENT OF REACTION BETWEEN REAGENT AND SAMPLE. TO OBTAIN AN ACCURATE MEASUREMENT OF THE EXTENT OF REACTION A REFERENCE ELECTRICAL SIGNAL IS REQUIRED. THIS IS PROVIDED IN THE PRESENT INVENTION BY ROTATING THE CUVET CONTAINING DISC AT A SPEED SUCH THAT AN INITIAL ELECTRICAL SIGNAL IS OBTAINED BEFORE THERE IS ANY SUBSTANTIAL REACTION BETWEEN REAGENT AND SAMPLE IN THE CUVETS.

H. N. HILL. JR

ANALYTICAL PROCESS Sept. 18, 1973 2 Sheets-Sheet 1 Filed Dec. 9, 1971 so 37 W INVENTOR F 6'. He er? N. Hill Jr.

%g/7wg ATTORNEY H. N. HILL. JR

ANALYTICAL PROCESS Sept. 18,1973":

2 Sheets-Sheet 2 Filed Dec. 9, 1971 5252mm 20:52am e25; l|||||||||l||J ww 5E5 :2: 220586 Eozms ME N 296555 mEzEu 13.3w :2: 02058 5 565: i v v Oh mm 5Ew z8 565 E @o z mm 6 ow M moBmEQ mm .Es W 555320611 Emu QEItEBS v m m k 22 IIL w INVENTOR Herbert N. Hill Jr.

$4M flay/ ATTORNEY United States Patent U.S. Cl. 23230 B 1 Claim ABSTRACT OF THE DISCLOSURE Known rotating spectrophotometers have a series of cuvets arranged concentrically around a horizontally rotatable disc so that when the disc is rotated, centrifugal force mixes and transfers reagents and samples to the cuvets. As each cuvet passes a light source, the absorbance of each individual sample is detected and measured photometrically and an electrical signal provided which is indicative of the extent of reaction between reagent and sample. To obtain an accurate measurement of the extent of reaction a reference electrical signal is required. This is provided in the present invention by rotating the cuvet containing disc at a speed such that an initial electrical signal is obtained before there is any substantial reaction between reagent and sample in the cuvets.

This invention relates to a method for rapidly operating a spectrophotometer adapted to analyze the presence of a substance in a plurality of samples which are rotating in a centrifugal field.

In recent years, the need for rapid, automatic analytical devices has increased markedly, owing to the numerous microanalytical studies in biochemical research, routine clinical testing for physicians and hospitals, enzymatic studies, and the like. However, few devices are available which can analyze sufiiciently rapidly and accurately to handle the increasing number of varied tests desired by clinicians and research workers.

Recently, multistation analytical photometers which utilize a centrifugal field have become available for the rapid microanalysis of a wide variety of liquids such as blood serum and other body fluids, food products, and the like. Since numerous analyses can be performed rapidly and simultaneously, these devices are of particular interest wherein a large number of samples is involved or a variety of tests on one sample is desired. Moreover, since these devices allow the use of relatively small volumes of reagents, the use of expensive reagents can be minimized.

One such device which utilizes a centrifugal field in microanalytical studies is described in Analytical Biochemistry, 28, 545-562 (1969). This device employs the principle of double-beam spectrophotometry wherein ab sorbancies of a liquid sample and a reference solution are intercompared. The system is basically a series of cuvets arranged around the periphery of a rotor so that when it is spun, centrifugal force simultaneously mixes and transfers reagents and samples to the cuvets where an analysis is made spectrophotometrically. A sample loading disc is provided which contains rows of cavities arranged concentrically. In the afore-mentioned device serum samples to be analyzed are placed in the inner cavities, and the reagents are placed in cavities at a greater radial distance than those containing the serum samples. The sample loading disc is then indexed and positioned in the rotor and as the rotor is accelerated, centrifugal force moves the sample to the cavity containing the reagent, where they are mixed and the mixture of reagent and sample is then moved through a communicating passage into the cuvet. The filled cuvets rapidly spin past a fixed light beam, and the transmission of light through the cuvets, i.e., through the samples, is measured.

In the past, when a variety of tests was to be performed,

3,759,666 Patented Sept. 18, 1973 a reagent blank, was provided for each sample-serum mixture to establish a zero absorbancy value for comparison with color changes during serum-reagent reaction whereby the extent of reaction between serum and reagent was established. This required that serum samples be placed in alternate cavities only, thus limiting the number of tests that could be performed at one time and the speed with which such tests could be made.

It is therefore an object of the present invention to provide a method for increasing the testing capability of rotating spectrophotometers employed for analytical testmg.

Other objects will be apparent from the following description and claim taken in conjunction with the draw ing wherein FIG. 1 shows a plan view of the rotatable disc of a spectrophotometer used in analytical testing FIG. 2 is an elevation view of the device of FIG. 1 also showing a light source, photodetection means, cuvet assembly and motor drive used in connection with the device of FIG. 1, and FIG. 3 shows schematically an electrical arrangement used in conjunction with the device of FIGS. 1 and 2.

The present invention will be more fully understood by reference to the drawing. With reference to FIGS. 1 and 2 there is shown a rotatable loading disc of a spectrophotometer of the type previously described which comprises thirty rows of cavities numbered 1 to 30 with each row having a serum cavity 33 and a reagent cavity 31. A slot to enable indexing of the loading disc is shown at 40. In the past, standard operating procedure was to place reagent in the cavities 31 in alternate positions e.g. 1, 3, 5 etc. through 29 with the adjacent radially aligned serum cavities 33 being left empty. Both serum and reagent were placed in their respective cavities 33 and 31 in positions 2, 4, 6, 8 etc. through 30. Thus a reagent blank was provided for each analysis to be performed in positions 1, 3, 5 etc. through 20. With the sample-reagent containing disc indexed and positioned in rotor assembly 35 each row of cavities was aligned with a respective cu-vette 37 and as the rotor assembly 35 was driven, centrifugal force moved the contents of the serum and reagents to the outermost cavities 39. Serum and reagents from positions 2, 4, 6, 8 etc. were mixed and transferred from cavity 39 through channels 50 to their respective cuvettes 37. The cuvettes communicating with the cavities of positions 1, 3, 5 etc. were filled only with reagent while the cuvettes 37 communicating with the cavities of positions 2, 4, 6, etc. were filled with a mixture of serum and reagent. The filled cuvettes 37 rotated between light source 55 and photomultiplier 60. The cuvettes 37 for positions 1, 3, 5 etc. contained only reagent, and photomultiplier 60 therefore provided a value which could be designated as zero absorbance i.e. a blank. The signals provided by the photomultiplier for the other positions 2, 4, 6 etc. could be compared to its corresponding blank thereby indicating any color changes due to reaction between reagent and serum.

Based on the observation that serum-reagent reactions require a finite period of time to exhibit a significant color change, i.e. a change in absorbance, a method is provided in the present invention whereby all positions 1 through 30 are provided with both serum and reagent in the respectve cavities 33 and 31. The disc 100 is then rotated very rapidly e.g. 1000 r.p.m. whereby all cuvettes are passed between light source 55 and photomultiplier 60 in a very short period of time e.g. less than A of a second. Thus, the signals provided by photomultiplier 60 during the initial rotation correspond to a blank since no significant color change due to serum-reagent reaction occurs in this interval. The electrical current signals from photomultiplier 60, with reference to FIG. 3, are applied to a conventional logarithmic amplifier 61. These signals are in the form of pulses of electrical current due to the chopping effect of the rotor assembly 35. A logarithmic amplifier is desirable due to the inherent logarithmic character of the absorbance phenomenon of serum-reagent reactions. In amplifier 61 the pulses of electrical current generated by photo-multiplier 60 are converted to pulses of voltage having a logarithmic relationship to the current pulses. The voltage pulses from logarithmic amplifier 60 are applied to a conventional peak detector 62 which can be a Peak Detector Module 4084/25 available from the Burr-Brown Research Corporation. The peak detector 62 observes the peak value of each pulse applied and holds this value until re-set by timing function generator 64 which is synchronized with and coupled to rotor assembly 35. Thus, during the initial rotation of rotor assembly 35, peak signals are applied to peak detector 62 for each of the cuvet positions 1 through 30, with the timing function generator 64 re-setting the peak detector 62 after each pulse is received. Each peak signal received by peak detector 62 is applied to analog-to-digital converter 66 which generates a group of pulses, using the conventional binary number system, having a value corresponding to the peak observed by the peak detector 62. The analogto-digital converter can be a Teledyne Philbrick Nexus model 4103. The pulse groups produced by analog-todigital converter 66 during the initial rotation of rotor assembly 35 are stored and held in memory unit 68 via electronic switch 70 which is actuated at the appropriate time by timing function generator 64. These pulse groups represent blanks since no significant reaction occurs between serum and reagent during the initial revolution of rotor assembly 35. The pulse groups produced by analog-to-digital converter 66 for subsequent revolutions of rotor assembly 35, at pre-selected times, are applied to memory unit 72 via electronic switch 74 which is actuated at the appropriate time by timing function generator 64.

When pulse groups are stored in memory unit 72, the contents of memory units 68 and 72 are subtracted in subtraction unit 75 and the resulting data is indicated at print-out unit 77, for example, a Moduprint Mode A available from Practical Automation Inc. This data is a measure of the color change or absorbance change in the cuvettes 37 of rotor assembly 35 and hence the extent of reaction between serum and reagent in the cuvettes.

By way of further illustration, a frequently performed analytical test is the determination of glucose in blood serum. In this analysis, microliters of serum is placed in serum cavities 33 and 350 microliters of glucose reagent by detecting changes in absorbance at a wavelength of 340 nm. An initial blank reading for this situation can be taken as much as two seconds after initiating the revolution of the rotor assembly 35 and stored in memory unit 68 to provide a blank. After a period of ten minutes a final reading is taken and stored in memory unit 72. The blank is subtracted from the value in memory unit 72 and the measured change is proportional to the change in absorbance due to the extent of oxidation of glucose which is concurrent with the reduction of NADP.

What is claimed is:

1. A method for determining the progress of a chemical reaction between serum samples and reagent which comprises (1) providing (a) a light source (b) a photodetector means spaced from and arranged in juxtaposition therewith, said photodetector means providing an electrical signal proportional to the optical density of the media by which it is separated from said light source and (c) a rotatably movable rotor assembly arranged to rotate between said light source and said photodetector means having (i) a plurality of light transmitting sample analysis chambers located at a common radial position in said rotor assembly and (ii) a reagent containing chamber and a serum containing chamber radially aligned with each of said sample analysis chambers whereby upon rotation of the rotor assembly the contents of the reagent and serum chambers are caused to mix and pass into the radially aligned sample analysis chambers (2) rotating said rotor assembly at a speed such that mixtures of serum and reagent enter the sample analysis chamber before any substantial reaction occurs therebetween (3) applying the electrical signal provided by said photodetector means at a time before any substantial reaction between reagent and serum occurs to a device adapted to store said electrical signal (4) applying an electrical signal provided by said photodetector means at a time after substantial reaction has occurred between reagent and serum to a device adapted to store said electrical signal (5) providing an electrical signal proportional to the difference between the two aforesaid electrical signals as a measure of the extent of reaction between serum and reagent.

References Cited UNITED STATES PATENTS 3,586,484 6/1971 Anderson 23-259 X 3,679,367 7/1972 Negersmith et al. 23-292 X 3,681,029 8/1972 Shapiro 23-259 MORRIS O. WOLK, Primary Examiner R. E. SERWIN, Assistant Examiner US. Cl. X.R. 

